KR20180050179A - Modified pd-l1 protein and use thereof - Google Patents

Abstract

The present invention relates to modified PD-L1 protein and a use thereof, and more specifically, the present invention provides Fc fusion protein comprising protein in which some of amino acid residues of the extracellular domain of PD-L1 are substituted with cysteine, and modified PD-L1 protein. The modified PD-L1 protein according to the present invention can be used for the prevention and treatment of cancer diseases, autoimmune diseases, inflammatory bowel disease, colitis, psoriasis, asthma, or arthritis.

Description

MODIFIED PD-Ll PROTEIN AND USE THEREOF < RTI ID = 0.0 >

The present invention relates to a modified PD-L1 protein with improved stability and activity. Specifically, it relates to PD-L1 having improved stability and activity by substituting cysteine with some of the amino acid residues of the extracellular domain of PD-L1 so as to form a dimer.

Human hPD-L1 (human Programmed Cell Death-Ligand 1) is a ligand for PD-1 (programmed death-1) as well as hematopoietic cells such as T lymphocytes, B lymphocytes, Dendritic cells, It is a type 1 transmembrane protein that is also expressed in non-hematopoietic cells such as cells, pancreatic cells, and hepatocytes.

In order to activate T cells, it is necessary to co-stimulate the T cell receptor in addition to the primary signal stimulation of the antigen and the antigen. At this time, if either signal is not present, the T cell becomes an anergy state. (PD-1) is an immune checkpoint or immune modulator that regulates T-cell secondary signaling activity. It activates activated T cells (CD8 and / or CD4) and dendritic cells (PD-L1) or B7.1 (CD80), which are expressed on the surface of cells, such as dendritic cells, by inhibiting T cell proliferation and decreasing cytokine expression. It can act to inhibit.

(Immunol Rev. 2010; 236: 219-42), the binding of PD-L1 to PD-L1 is known to induce the activity of regulatory T cells (Rheumatol Int. 2011 Apr; 31 (4) (1994)), when PD-L1-Ig fusion protein (PD-L1-Ig) was injected into a CIA (collagen-induced arthritis) mouse model : 513-9). Since PD-1 is expressed in activated T cells, PD-L1 protein is expected to be useful not only for autoimmune diseases but also as a therapeutic agent specifically targeting active immune cells in inducing immune tolerance in organ transplantation .

To date, therapeutic agents for the PD-1 / PD-L1 cell signaling system have been developed as an antagonist to increase tolerance to T cell activation by tolerance breaking. However, immunotherapeutic agents based on induction of T cell immunity using an agonist have not been developed until now. The PD-1 / PD-L1 agonist, which can be easily developed using antibody fusion techniques in the case of PD-1 / PD-L1 antagonists, should be developed as a soluble form of the protein It is not technically easy to develop.

The present invention relates to a modified PD-L1 protein in which part of the amino acid residues of the sequence of Ig-V-like domain and / or Ig-C-like domain of the PD-L1 protein is substituted with cysteine .

In order to solve this problem, the present invention is a modified PD-L1 protein, which comprises an Ig-V-like domain of a human PD-L1 protein, wherein the Ig- 26, and 72 to 76 amino acid residues are substituted with cysteine.

The modified PD-L1 protein comprises an Ig-V-like domain and an Ig-C-like domain, wherein the modified PD-L1 protein comprises the amino acid sequence of SEQ ID NO: 1 from 22 to 25, 36 to 44, 94, 136 to 145, 154, 185, 196 and 228 to 230 amino acid residues are substituted with cysteine.

In addition, it provides a modified PD-L1 protein in the form of a dimer in which two fusion proteins with different sequences are bound.

The modified PD-L1 protein according to the present invention is characterized in that a part of the amino acid residues of the sequence of the human PD-L1 protein is substituted with cysteine, and a stable dimer can be formed by the reaction of the substituted cysteine amino acid residue . In addition, the modified dimer of PD-L1 has a characteristic that it can bind to the PD-L1 receptor more effectively than the monomer. Therefore, when the modified protein of the present invention is used as an antagonist or an inhibitor of the signal transduction system of PD-L1, it is possible to provide pharmaceuticals which can prevent and treat diseases such as cancer diseases, autoimmune diseases, inflammatory diseases and graft- Composition, or immune tolerance inducer. The present invention also relates to a composition for preventing and / or treating an immune tolerance.

FIGS. 1A through 1E illustrate Fc fusion proteins comprising a modified PD-L1 protein dimer and a dimer according to an embodiment of the present invention.
2 shows a fusion protein (PD-L1_V) comprising a PD-L1 dimer in which an amino acid residue of an Ig-V-like domain is substituted with cysteine.
Fig. 3 shows a fusion protein (PD-L1_C) comprising a PD-L1 dimer in which the amino acid residue of the Ig-C-like domain is substituted with cysteine.
Fig. 4 shows a fusion protein (PD-L1_VC) comprising a PD-L1 dimer in which the amino acid residues of Ig-V-like domain and Ig-C-like domain are substituted with cysteine.
FIG. 5 shows the results of SDS-PAGE analysis for purifying purified proteins and PD-L1_V, PD-L1_C and PD-L1_VC proteins.
6A is a result of performing SE-HPLC analysis on the PD-L1_V protein.
6B is a result of performing SE-HPLC analysis on the PD-L1_C protein.
Fig. 6C shows the result of performing SE-HPLC analysis on the PD-L1_VC protein.
FIG. 7 shows the results of confirming human T cell proliferation (T cell proliferation) inhibitory ability using PD-L1_V, PD-L1_C and PD-L1_VC proteins.
FIG. 8 shows the results of confirming the inhibitory effect of human-derived T cells on the production of inflammatory cytokines (IFNr) using PD-L1_V, PD-L1_C and PD-L1_VC proteins.

V-like domain of the human PD-L1 protein, wherein the Ig-V-like domain comprises the amino acid sequence of SEQ ID NO: 2, 4 to 7, 18, 26, and 72 to 76 amino acid residues are substituted with cysteine.

The extracellular domain of PD-L1 may be a polypeptide comprising the Ig-V-like domain of PD-L1.

Specifically, the extracellular domain of PD-L1 may be a polypeptide consisting of amino acids 19 to 238 of SEQ ID NO: 1 or a polypeptide consisting of amino acids 19 to 239 of SEQ ID NO: 1.

At this time, the extracellular domain of PD-L1 contains an Ig-V-like (Ig V, Ig V) sequence, which is a conserved sequence similar to the amino acid sequence of an immunoglobulin, The Ig-V-like sequence is the amino acid sequence of SEQ ID NO: 1, 68 to 114. Also, a highly conserved sequence region containing Ig-C-like (Ig C, Ig C like) sequence is an amino acid sequence 153 to 210 of SEQ ID NO: 1. In addition, the fragment of the PD-L1 extracellular domain may comprise all or part of an Ig-V-like domain, including an Ig-V-like sequence of PD-L1.

In addition, the extracellular domain of PD-L1 or a fragment thereof may be derived from human or mouse. Specifically, the human PD-L1 protein has 290 amino acid residues and has an amino acid sequence represented by SEQ ID NO: 1 (Accession Number: Q9NZQ7). In the amino acid sequence of SEQ ID NO: 1, the amino acid residues 1 to 18 at the N-terminus are signal sequences. The mature human PD-L1 is a protein consisting of amino acids 19-290 of SEQ ID NO: 1. The extracellular domain of human PD-L1 comprises amino acids 19 to 238 of SEQ ID NO: 1 or 19 to 239 of SEQ ID NO: 1. The human PD-L1 protein comprises an Ig-V-like domain which is amino acids 19 to 127 of SEQ ID NO: 1 and an Ig-C-like domain which is an amino acid from 134 to 226 or 133 to 225 of SEQ ID NO:

The extracellular domain of PD-L1 may include a polypeptide consisting of the 19th to 239th amino acid sequence of SEQ ID NO: 1. The extracellular domain of PD-L1 may be one in which at least one amino acid at positions corresponding to 22 to 25, 36 to 44, 90 to 94, 136 to 145 of SEQ ID NO: 1 is substituted with cysteine. In addition, the extracellular domain of PD-L1 has about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or more of the polypeptide sequence consisting of amino acid sequence 19 to 239 of SEQ ID NO: , 94%, 95%, 96%, 97%, 98%, or 99% identical.

The Ig-V-like domain of the PD-L1 extracellular domain may be a polypeptide having the amino acid residues 21 to 114 of SEQ ID NO: 1 and capable of interacting with the receptor of PD-L1, Or a polypeptide having amino acids 19-114. Also, it may be a polypeptide having amino acids 21 to 120 of SEQ ID NO: 1, or a polypeptide having amino acids 19 to 120 of SEQ ID NO: 1. Also, the polypeptide may be a polypeptide having amino acids 21 to 127 of SEQ ID NO: 1 or a polypeptide having amino acids 19 to 127 of SEQ ID NO: 1 (SEQ ID NO: 4). Also, the polypeptide may be a polypeptide having amino acids 21 to 130 of SEQ ID NO: 1, or a polypeptide having amino acids 19 to 130 of SEQ ID NO: 1. Also, it may be a polypeptide having amino acids 21 to 131 of SEQ ID NO: 1, or a polypeptide having amino acids 19 to 131 of SEQ ID NO: 1. Also, the polypeptide may be a polypeptide having amino acids 21 to 133 of SEQ ID NO: 1, or a polypeptide having amino acids 19 to 133 of SEQ ID NO: 1 (SEQ ID NO: 2).

In addition, when the fragment of PD-L1 extracellular domain includes Ig-V-like domain or fragment thereof, it may further comprise an Ig-C-like domain of PD-L1 extracellular domain. The Ig-C-like domain may be a polypeptide having amino acids 133 to 225 of SEQ ID NO: 1, or a polypeptide having amino acids 134 to 226 of SEQ ID NO: 1.

In addition, when the fragment of the PD-L1 extracellular domain includes an Ig-V-like domain or a fragment thereof, it may further comprise a polypeptide comprising Ig-C-like domain of the PD-L1 extracellular domain or a fragment thereof . The polypeptide comprising the Ig-C-like domain may be a polypeptide having the amino acid numbers 134 to 239 of SEQ ID NO: 1 (SEQ ID NO: 5) or a polypeptide having the amino acid numbers 134 to 238 of SEQ ID NO: 1 (SEQ ID NO: have.

Also, the modified PD-L1 protein comprises an Ig-V-like domain and an Ig-C-like domain, wherein the modified PD-L1 protein comprises the amino acid sequence of SEQ ID NO: 1, 22-25, 36-44, 90-94, 136 to 145, 154, 185, 196 and 228 to 230 amino acid residues are substituted with cysteine.

In one embodiment of the PD-L1 extracellular domain fragment, the extracellular domain may comprise the entire Ig-V-like domain or a fragment thereof. In addition, the fragment of the PD-L1 extracellular domain may further comprise a polypeptide further comprising an Ig-C-like domain or an Ig-C-like domain.

The PD-L1 extracellular domain or fragment thereof may comprise various modified proteins or peptides. The modification may be performed by substituting, deleting or adding one or more proteins to the wild-type PD-L1, so long as the function of the PD-L1 is not altered. These various proteins or peptides can be used in combination with wild-type proteins such as 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, % ≪ / RTI > homology.

The term "PD-L1-Fc fusion protein" and "PD-L1-immunoglobulin Fc region fusion protein ", as used herein, refers to a fusion protein of PD-L1 protein, PD- ≪ / RTI > region. In this case, the PD-L1 protein may have a specific site of the Ig-V-like domain described above substituted with cysteine, and may further include an Ig-C-like domain or an Ig-C-like domain in which a specific site is substituted with cysteine have. Wherein the Fc region of the Fc region fusion protein of the Fc fusion protein or immunoglobulin comprises the CH2 domain and the CH3 domain in the C-terminal direction from the N-terminus. The Fc region may be any one of the Fc regions of IgG1, IgG2, IgD and IgG4, or a combination thereof (hybrid). The Fc region may also be a Fc region in which the C1q binding site and / or the Fc gamma receptor binding site and / or the FcRn binding site are modified.

On the other hand, the modified PD-L1 protein may include a linker. In a dimer containing only the modified Ig-V domain, the linker may be linked to the C-terminus of the first PD-L1 and the N-terminus of the second PD-L1. In a dimer that additionally contains an Ig-C or modified Ig-C domain in the modified Ig-V domain, the linker binds the C-terminus of the Ig-C region of the first PD-L1 and the Ig- Terminal of the < / RTI > The modified PD-L1 protein may be linked to the Fc region of the immunoglobulin by a linker or an Fc-hinge region. The linker connecting the immunoglobulin Fc region binds to the C-terminal or N-terminal of the modified PD-L1 protein. The linker can be linked to the free radical at the N-terminal or C-terminal end of the Fc fragment. In addition, the linker may be an albumin linker or a peptide linker. The linker may comprise 1 to 50 amino acids, 3 to 40 amino acids, and 10 to 30 amino acids. In addition, the peptide linker may be a peptide consisting of Gly and Ser residues. In addition, the peptide linker may be selected from the group consisting of leucine (Leu, L), isoleucine (Ile, I), alanine (Ala, A), valine (Val, V), proline A peptide consisting of 1 to 10 amino acids selected from the group consisting of Arg, R, Asn, N, Ser, S and Gln, Q.

In addition, the linker may be a polypeptide consisting of 3 to 15 amino acids composed of glycine (Gly, G) and serine (Ser, S) residues, and may be composed of 6 to 11 amino acids. In one embodiment of the invention, the linker may comprise an amino acid sequence of GSGGGS or GSGGGGSGGGS.

Also, the combined or hybrid immunoglobulin Fc region comprises a CH2 domain and a CH3 domain, wherein the CH2 domain comprises a portion of an amino acid residue of a human IgD and a CH2 domain of a human IgG4, wherein the CH3 domain is a human IgG4 (I), (I '), (Ia), (I') and (I ') of the combined or hybrid immunoglobulin Fc region -a), (Ib) and (I'-b)) can be any of the Fc regions of IgG1, IgG2, IgG3, IgD and IgG4 or a combination thereof.

The Fc region is modified so that binding and / or complement binding does not occur with the Fc receptor. In particular, the Fc region of the modified immunoglobulin comprises a CH2 domain and a CH3 domain in the N-terminal to C-terminal direction, wherein the CH2 domain comprises a portion of the amino acid residue of the CH2 domain of human IgD and human IgG4, The CH3 domain may comprise a portion of the amino acid residue of the CH3 domain of human IgG4.

As used herein, the term "Fc region", "Fc fragment" or "Fc" refers to the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, Region and light chain constant region 1 (CL1). It may further comprise a hinge region of the heavy chain constant region. Hybrid Fc or hybrid Fc fragments may also be referred to herein as "hFc" or "hyFc". As used herein, the term "Fc region variant" means that some amino acids in the Fc region are substituted or produced by combining different Fc regions. The Fc region variant may be modified to prevent cleavage at the hinge region.

In addition, the Fc region or Fc region variant of the modified immunoglobulin can be represented by the following formula:

N '- (Z1) p-Y-Z2-Z3-Z4-C'

Wherein N 'is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide;

p is an integer of 0 or 1; And

Z1 is an amino acid sequence having 5 to 9 consecutive amino acid residues in the N-terminal direction from the 98 position in the amino acid residues at positions 90 to 98 of SEQ ID NO: 7,

Y is an amino acid sequence having 5 to 64 consecutive amino acid residues in the N-terminal direction from the 162 position in the amino acid residues 99 to 162 of SEQ ID NO: 7,

Z2 is an amino acid sequence having 4 to 37 consecutive amino acid residues in the C-terminal direction from the 163 position in the amino acid residue at positions 163 to 199 in SEQ ID NO: 7,

Z3 is an amino acid sequence having 71 to 106 contiguous amino acid residues in the N-terminal direction from the 220 position in the amino acid residues 115 to 220 of SEQ ID NO: 8, and

Z4 is an amino acid sequence having 80 to 107 amino acid sequences from the 221 position to the C-terminal direction in the amino acid residues 221 to 327 of SEQ ID NO: 8.

The modified Ig Fc domain may be as described in U.S. Patent No. 7,867,491 and the production of the modified Ig Fc domain may be performed with reference to what is described in U.S. Patent No. 7,867,491.

In addition, the Fc fragment of the present invention may be a natural type sugar chain, an increased sugar chain compared to the native type, a reduced sugar chain compared to the native type, or a form in which the sugar chain is removed. Increase, decrease or elimination of immunoglobulin Fc sugar chains can be performed by conventional methods known in the art such as chemical methods, enzymatic methods and genetic engineering engineering methods using microorganisms. Removal of the sugar chain from the Fc fragment rapidly reduces the binding affinity of the primary complement component C1 to C1q and results in a decrease or loss of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) Thereby not inducing unnecessary immune responses in vivo. In this regard, immunoglobulin Fc fragments in the deglycosylated and aglycosylated form may be more suitable for the purposes of the present invention as carriers of the drug. As used herein, the term " deglycosylation "means enzymatically eliminating sugar from an Fc fragment, and the term" aglycosylation "means that the Fc fragment is expressed in a prokaryote, preferably E. coli Which means that it is produced in an unglycosylated form.

The Fc region of the modified immunoglobulin includes the amino acid sequence of SEQ ID NO: 9 (hyFc), SEQ ID NO: 10 (hyFcM1), SEQ ID NO: 11 (hyFcM2), SEQ ID NO: 12 (hyFcM3) can do.

In another aspect of the present invention, there is provided a dimeric fusion protein comprising a PD-L1 protein, wherein the dimeric fusion protein comprises a first monomer and a second monomer selected from the above-mentioned fusion proteins, wherein the first monomer and the second monomer have different sequences And a fusion protein in which any one of the cysteine residues of the first monomer and the cysteine residue of the second monomer is covalently bonded can be provided. In addition, the covalent bond may be a disulfide bond.

The fusion protein comprising the extracellular domain of PD-L1 according to the present invention is characterized in that at least one of the amino acid residues at a specific position is substituted with cysteine, and the fusion protein includes two of such fusion protein monomers, A covalent bond can be made so that a fusion protein of a stable heterodimer can be provided.

When the cysteine in the amino acid sequence forming the beta-sheet of the first monomer and the cysteine in the amino acid sequence forming the beta-sheet of the second monomer are located at a distance of about 7, 5, 4 or 3 or less, A disulfide bond is formed between the fusion protein and the fusion protein.

The heterodimer fusion protein according to one embodiment of the present invention is characterized in that any one of the amino acid residues at positions 72 to 76 of SEQ ID NO: 2 of the lg-V-like domain of the first monomer and the lg- At least one of the amino acid residues at positions 4 to 7 of SEQ ID NO: 2 in the domain may be cysteine.

Specifically, the amino acid residue at position 76 of SEQ ID NO: 2 of the first monomeric lg-V-like domain and the amino acid residue at position 7 of SEQ ID NO: 2 of the lg-V-like domain of the second monomer may be cysteine. The amino acid residue at position 23 of SEQ ID NO: 2 of the first monomeric lg-V-like domain and the amino acid residue at position 26 of SEQ ID NO: 2 of the lg-V-like domain of the second monomer may be cysteine. The amino acid residue at position 73 of SEQ ID NO: 2 of the first monomer lg-V-like domain and the amino acid residue at position 4 of SEQ ID NO: 2 of the lg-V-like domain of the second monomer may be cysteine. The amino acid residue at position 21 in SEQ ID NO: 2 of the first monomer lg-V-like domain and the amino acid residue at position 23 of SEQ ID NO: 2 in the lg-V-like domain of the second monomer may be cysteine. The amino acid residue at position 76 of SEQ ID NO: 2 of the first monomeric lg-V-like domain and the amino acid residue at position 6 of SEQ ID NO: 2 of the lg-V-like domain of the second monomer may be cysteine.

The amino acid residues 136-140, 154, and 185 and 196 of SEQ ID NO: 1 of the PD-L1 protein of the first monomer and the amino acids 139-143 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer At least one of the residues may be cysteine. Specifically, the amino acid residue at position 136 of SEQ ID NO: 1 of the PD-L1 protein of the first monomer and the amino acid residue at position 139 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer may be cysteine. In addition, the amino acid residue at position 137 of SEQ ID NO: 1 of PD-L1 protein of the first monomer and the amino acid residue at position 140 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer may be cysteine. In addition, the amino acid residue at position 138 of SEQ ID NO: 1 of the PD-L1 protein of the first monomer and the amino acid residue at position 141 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer may be cysteine. The amino acid residue at position 139 of SEQ ID NO: 1 of the PD-L1 protein of the first monomer and the amino acid residue at position 142 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer may be cysteine. The amino acid residue at position 140 of SEQ ID NO: 1 of the PD-L1 protein of the first monomer and the amino acid residue at position 143 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer may be cysteine. The amino acid residue at position 154 of SEQ ID NO: 1 of PD-L1 protein of the first monomer and the amino acid residue at position 229 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer may be cysteine. The amino acid residue at position 196 of SEQ ID NO: 1 of the PD-L1 protein of the first monomer and the amino acid residue at position 230 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer may be cysteine. The amino acid residue at position 185 of SEQ ID NO: 1 of PD-L1 protein of the first monomer and the amino acid residue at position 228 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer may be cysteine.

In addition, the amino acid residues at positions 94 and 136 of SEQ ID NO: 1 of the PD-L1 protein of the first monomer and the amino acid residues at positions 25 and 139 of SEQ ID NO: 1 of the PD-L1 protein of the second monomer may be cysteine .

In addition, one of the CH3 domains of the Fc region of the modified monomer of the first monomer and the modified monomer of the second monomer may be a knob modification, and the other may include a hole modification. Thus, the first monomer and the second monomer form a knob-into-hole pair, so that the fusion protein can be realized with a more stable heterodimer structure.

Specifically, the knob modification may be such that the amino acid residue at position 152 of SEQ ID NO: 9 is cysteine and the amino acid residue at position 164 is tryptophan. In addition, the hole modification may be such that the amino acid residue at position 147 of SEQ ID NO: 9 is cysteine, the amino acid residue at position 164 is serine, the amino acid residue at position 166 is alanine, and the amino acid residue at position 205 is valine.

Another aspect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of immune diseases comprising a modified PD-L1 protein.

The immunological disease may be a disease selected from the group consisting of autoimmune diseases, inflammatory diseases, and transplantation rejection diseases of cells, tissues, or organs.

Another aspect of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer diseases comprising a modified PD-L1 protein.

Yet another aspect of the present invention is to provide a composition for inducing an immunotolerance comprising a dimeric fusion protein consisting of an extracellular domain of PD-L1 and an Fc region of a modified immunoglobulin. As used herein, the term "immune tolerance" refers to a condition in which the immune system does not exhibit a tissue destruction response to a particular antigen while not being in a persistent immunosuppressive state. In particular, the fusion protein may be used for immune tolerance where regulatory T cells are involved. Preferably, the fusion protein can be used to inhibit an immune response that occurs upon tissue grafting.

In addition, the fusion protein comprising the PD-L1 extracellular domain or a fragment thereof can be administered to a subject in a variety of ways. For example, pharmacological compositions can be administered parenterally, examples being subcutaneous, intramuscular, or intravenous.

Another aspect of the present invention provides a method for treating a disease by administering a composition comprising the PD-L1 fusion protein of the present invention as a pharmacologically active ingredient.

Such methods include administering an effective amount of a PD-L1 fusion protein dimer of the invention to a mammal having a health condition that is directly or indirectly associated with the intended disease. In addition, the PD-L1 fusion protein dimer may be administered to a subject, preferably a mammal, including a human, in a therapeutically effective amount. The chimeric polypeptide may be administered by intravenous, subcutaneous, oral, buccal, sublingual, nasal, parenteral, rectal, vaginal or pulmonary routes.

The unit dose of the fusion protein dimer of the present invention is 0.1 mg / kg to 1,500 mg / kg in humans. In one embodiment, the unit dose of the fusion protein is from 1 mg / kg to 100 mg / kg in humans. In yet another embodiment, the unit dose of the fusion protein is 5 mg / kg to 20 mg / kg in humans. The unit dose may vary depending on the disease to be treated and the presence or absence of side effects. However, the optimal dosage can be determined using routine experimentation. The administration of the fusion protein can be effected either by periodic bolus injections or from an external reservoir (e.g., intravenous bag) or medial (e.g., bioerodable implant) Intravenous, subcutaneous, or intraperitoneal administration.

The compositions of the present invention may be administered in combination with other drugs or physiologically active substances having a preventive or therapeutic effect for a disease to be prevented or treated or may be formulated in the form of a combination formulation with such other drugs .

The method for preventing or treating disease using the fusion protein or composition of the present invention may include administration of another drug or physiologically active substance having the effect of preventing or treating disease using the fusion protein or composition of the present invention The route of administration, the timing of administration, and the administration dose may be determined depending on the type of the disease, the disease state of the patient, the purpose of treatment or prevention, and other drugs or physiologically active substances used in combination.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  1. PD- L1 _V, PD- L1 _C and PD- L1 _VC gene Construct  Produce

In order to derive a dimer of PD-L1_V, Leucine 94 was substituted with cysteine in the V domain of PD-L1-1, and the V domain of PD-L1-2 was substituted with cysteine to form PD-L1-1 Of substituted cysteine 94 and PD-L1-2 substituted cysteine 25 were able to form a disulfide bond. In order to derive the dimer of PD-L1_C, lysine 136 was substituted with cysteine in the C domain of PD-L1-1, and the C domain of PD-L1-2 was replaced with cysteine, Substituted cysteine 136 of PD-L1-2 and 139 of substituted cysteine of PD-L1-2 were able to form a disulfide bond. In order to derive a dimer of PD-L1_VC, Leucine 94 was substituted with cysteine in the V domain of PD-L1-1, lysine 136 was substituted with cysteine in the C domain of PD-L1-1, PD- 2, the lysine 25 was substituted with cysteine, and the C domain of PD-L1-2 replaced glutamine 139 with cysteine. Substituted cysteine 94 of PD-L1-1 and substituted cysteine 25 of PD-L1-2 were allowed to form a disulfide bond, and the substituted cysteine 136 of PD-L1-1 and PD-L1-2 The substituted cysteine 139 was allowed to form a disulfide bond. For constructing PD-L1_V, PD-L1_C and PD-L1_VC, constructs were constructed by fusing PD-L1 to the N-terminus of immunoglobulin Fc in order to increase the half-life of the system.

Specifically, the human PD-L1 gene, which is a known amino acid sequence (accession number: Q9NZQ7), was used to produce an expression vector containing the human PD-L1 structural gene. In addition, a construct containing only the extracellular domain was prepared and fused to the C-terminal of the modified Fc domain to obtain a recombinant protein expression vector.

PD-L1_V (P1-C) and PD-L1_VC (P1-V) were used to construct the PD-L1_V-Fc, PD-L1_C- VC) were synthesized from TOP Gene Technologies (Canada, Quebec). Overlapping PCR was performed to prepare two gene fragments as one gene fragment. HyFc-hPD-L1 (V + C or C domain) was inserted into the pTrans vector by treatment with restriction enzymes KpnI-HF (R3142, NEB) and NotI-HF (R3189, NEB).

For the modified Fc domain, it is described in US Patent 7,867,491 as hyFc (Hybrid Fc). The hyFc protein is a hybrid type of Fc of human IgD and Fc of human IgG4 and can exhibit an excellent half-life of the body when bound to a bioactive protein as compared with the Fc region of a previously modified immunoglobulin. Among the various kinds of hyFc proteins, a recombinant protein expression vector was prepared using the amino acid sequence (hyFc) of SEQ ID NO: 6 in this experiment.

The hPD-L1 (V + C) and hPD-L1 (V) forms, which contain all the PD-L1 extracellular domains (19-239 aa) and the V domain of 19-133aa , A recombinant expression vector containing a gene fused to the N-terminal of hyFc was constructed using a novel linker (Linker 1) derived from IgD hinge. The prepared expression vector was transfected with Expi CHO-S transient transfection method and cultured for 7 days, and the productivity of each culture was confirmed from the obtained culture.

Example  2. PD- L1 _V, PD- L1 _C and PD- L1 Securing _VC protein

The expression levels of PD-L1_V-Fc, PD-L1_C-Fc and PD-L1_VC-Fc proteins produced using the Expi CHO-S production system of Example 1 were measured by enzyme immunoassay (Human IgG Quantitation Kit, E80-104). In order to purify the proteins of PD-L1_V-Fc, PD-L1_C-Fc and PD-L1_VC-Fc from the culture medium, impurities other than Fc were removed through Protein A resin, and aggregation and truncation types were removed through Q Sepharose FF resin .

The target protein was eluted with elution buffer (0.1 M glycine, pH 3.0) through Protein A resin, and the eluate was immediately neutralized to neutral pH using 1 M Tris. The neutralized eluate was purified by SDS-PAGE and SE-HPLC (FIG. 5 and FIG. 6) by recovering pure target protein only by elution with a gradient of salt concentration using Q Separose FF resin.

The purified PD-L1_V-Fc, PD-L1_C-Fc and PD-L1_VC-Fc proteins were identified by SDS-PAGE analysis and found to be between about 100 kDa and 150 kDa in the non-reducing condition. And the size marker was confirmed to be about 50 ~ 75kDa in the reducing condition. The purity of the PD-L1_V-Fc was 91.3%, the purity of the PD-L1_C-Fc was 88.3%, and the PD-L1_VC-Fc was 88.5% 6a, 6b, 6c)

Example  3. PD- L1 _V ( P1 -V), PD- L1 _C ( P1 -C) and PD- L1 _VC ( P1 -VC) protein in vitro - T cell proliferation Inhibition  compare

In order to confirm the in vitro activity using the PD-L1_V, PD-L1_C and PD-L1_VC proteins obtained in Example 2, human PB-1 cells were cultured in PBMC of healthy adult blood using human CD4 + -VC < / RTI > inhibition of T cell proliferation was compared in vitro. The plate was coated with 1 μg / ml of anti-CD3 (OKT3) and 0.5 μM of P1-V, P1-C and P1-VC fusion proteins on a 96-well plate.

In 96-well plates coated with anti-CD3 and P1-V, P1-C, and P1-VC fusion proteins, CD4 + T cells were stimulated in PBMC of healthy adult blood and 96 hours after the cell trace dilution of CD4 + PD1 + T cells Were analyzed by flow cytometry (FACS) and the effects of P1-V, P1-C, and P1-VC on cell proliferation were measured. As a negative control, 0.5 μM hyFc was used, and PDL1-IgG1-Fc and GX-P1 of the R & D system and tacrolimus, an immunosuppressant, were used as positive control groups at a concentration of 10 ng / ml.

As a result, P1-V, P1-C, and P1-VC confirmed the effect of inhibiting human CD4 + T cell proliferation (cell trace dilution reduction) (FIG. 7). These results confirm that P1-V, P1-C, and P1-VC have cell proliferation inhibitory ability in all of CD4 + T cells derived from 8 healthy adults.

Example  4. PD- L1 _V ( P1 -V), PD- L1 _C ( P1 -C) and PD- L1 _VC ( P1 -VC) Protein Expression of Inflammatory Cytokines

The efficacy of inhibiting inflammatory cytokines in human P1-V, P1-C, and P1-VC by using CD4 + T cells in healthy adult blood PBMCs was compared in vitro. The concentration of anti-CD3 was 1 ug / ml, and 0.5 μM of P1-V, P1-C, and P1-VC fusion proteins were coated on a 48-well plate. The CD4 + T cells were stimulated in PBMC of healthy adult blood in a 48-well plate coated with anti-CD3 and P1-V, P1-C and P1-VC fusion proteins, and the supernatant was obtained at 72 hours and the level of IFNr expression was measured by ELISA (R & D, DY285) analysis method. As a negative control, 0.5 μM hyFc was used. For the positive control, PDL1-IgG1-Fc and GX-P1 of the R & D system and tacrolimus, an immunosuppressant, were used at a concentration of 10 ng / ml.

As a result, it was confirmed that P1-V, P1-C, and P1-VC inhibited the expression of inflammatory cytokines secreted by CD4 + T cells (FIG. 8). From the above results, it was confirmed that the P1-V, P1-C, and P1-VC fusion proteins inhibit the proliferative capacity of human T-cell itself as well as suppress the expression of inflammatory cytokines, T cells in the presence of T cells.

<110> Genexine, Inc.          PROGEN CO., LTD. <120> MODIFIED PD-L1 PROTEIN AND USE THEREOF <130> FPD201610-0063C <160> 13 <170> Kopatentin 2.0 <210> 1 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of human PD-L1 (Accession number: Q9NZQ7) <400> 1 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu   1 5 10 15 Asn Ala Phe Thr Val Thr Val Lys Asp Leu Tyr Val Val Glu Tyr              20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu          35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile      50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser  65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn                  85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr             100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val         115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val     130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser                 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn             180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr         195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu     210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu                 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys             260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu         275 280 285 Glu Thr     290 <210> 2 <211> 115 <212> PRT <213> Artificial Sequence <220> Amino acid sequence of human PD-L1 V like domain (19-133) <400> 2 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser   1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu              20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln          35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg      50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala  65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys                  85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val             100 105 110 Asn Ala Pro         115 <210> 3 <211> 107 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > amino acid sequence of mouse PD-L1 V like domain (21-127) <400> 3 Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser Asn Val   1 5 10 15 Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu Asp Leu Leu Ala              20 25 30 Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val Ile Gln Phe Val          35 40 45 Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn Phe Arg Gly Arg      50 55 60 Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala Ala Leu Gln  65 70 75 80 Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Cys Cys Ile Ile                  85 90 95 Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr             100 105 <210> 4 <211> 109 <212> PRT <213> Artificial Sequence <220> Aamino acid sequence of human PD-L1 V like domain (19-127) <400> 4 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser   1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu              20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln          35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg      50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala  65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys                  85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr             100 105 <210> 5 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence containing human PD-L1 C-like domain (134-239) <400> 5 Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro Val Thr Ser   1 5 10 15 Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala Glu Val              20 25 30 Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys Thr Thr Thr          35 40 45 Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr Ser Thr Leu      50 55 60 Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe Arg Arg  65 70 75 80 Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile Pro Glu Leu                  85 90 95 Pro Leu Ala His Pro Pro Asn Glu Arg Thr             100 105 <210> 6 <211> 105 <212> PRT <213> Artificial Sequence <220> Amino acid sequence including human PD-L1 C like domain (134-238) <400> 6 Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro Val Thr Ser   1 5 10 15 Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys Ala Glu Val              20 25 30 Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys Thr Thr Thr          35 40 45 Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr Ser Thr Leu      50 55 60 Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr Phe Arg Arg  65 70 75 80 Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile Pro Glu Leu                  85 90 95 Pro Leu Ala His Pro Pro Asn Glu Arg             100 105 <210> 7 <211> 384 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of human IgD constant region (Genbank          accession No. P01880) <400> 7 Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Ile Ile Ser Gly Cys Arg   1 5 10 15 His Pro Lys Asp Asn Ser Pro Val Val Leu Ala Cys Leu Ile Thr Gly              20 25 30 Tyr His Pro Thr Ser Val Thr Val Thr Trp Tyr Met Gly Thr Gln Ser          35 40 45 Gln Pro Gln Arg Thr Phe Pro Glu Ile Gln Arg Arg Asp Ser Tyr Tyr      50 55 60 Met Thr Ser Ser Gln Leu Ser Thr Pro Leu Gln Gln Trp Arg Gln Gly  65 70 75 80 Glu Tyr Lys Cys Val Val Gln His Thr Ala Ser Lys Ser Lys Lys Glu                  85 90 95 Ile Phe Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro             100 105 110 Thr Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala         115 120 125 Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys     130 135 140 Glu Lys Glu Lys Glu Glu Glu Glu Glu Arg Glu Thr Lys Thr Pro Glu 145 150 155 160 Cys Pro Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala                 165 170 175 Val Gln Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val             180 185 190 Val Gly Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly         195 200 205 Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser     210 215 220 Asn Gly Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu 225 230 235 240 Trp Asn Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu                 245 250 255 Pro Pro Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro             260 265 270 Val Lys Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala         275 280 285 Ala Ser Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile     290 295 300 Leu Leu Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe 305 310 315 320 Ala Pro Ala Arg Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala                 325 330 335 Trp Ser Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr             340 345 350 Tyr Thr Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala         355 360 365 Ser Arg Ser Leu Glu Val Ser Tyr Val Thr Asp His Gly Pro Met Lys     370 375 380 <210> 8 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Partial human IgG4 constant region          (Genbank accession No. AAH25985) <400> 8 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg   1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr              20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser          35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser      50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr  65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                  85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro             100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys         115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val     130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe                 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp             180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu         195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg     210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp                 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys             260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser         275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser     290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys                 325 <210> 9 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hyFc <400> 9 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys   1 5 10 15 Glu Glu Glu Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His              20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr          35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val      50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val  65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser                  85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu             100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser         115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro     130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala                 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr             180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu         195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser     210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys                 245 <210> 10 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hyFcM1 <400> 10 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Gly Gly Lys Glu Lys   1 5 10 15 Glu Glu Glu Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His              20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr          35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val      50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val  65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser                  85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu             100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser         115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro     130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala                 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr             180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu         195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser     210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys                 245 <210> 11 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hyFcM2 <400> 11 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Gly Ser Lys Glu Lys   1 5 10 15 Glu Glu Glu Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His              20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr          35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val      50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val  65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser                  85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu             100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser         115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro     130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala                 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr             180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu         195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser     210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys                 245 <210> 12 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hyFcM3 <400> 12 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Ser Gly Lys Glu Lys   1 5 10 15 Glu Glu Glu Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His              20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr          35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val      50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val  65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser                  85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu             100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser         115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro     130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala                 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr             180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu         195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser     210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys                 245 <210> 13 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of hyFcM4 <400> 13 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Ser Ser Lys Glu Lys   1 5 10 15 Glu Glu Glu Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His              20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr          35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val      50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val  65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser                  85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu             100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser         115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro     130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala                 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr             180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu         195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser     210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys                 245

Claims (18)

As the PD-L1 protein,
The PD-L1 protein comprises an Ig-V-like domain of a human PD-L1 protein,
The human Ig-V-like domain is SEQ ID NO: 2,
Wherein at least one of the amino acid residues at positions 4 to 7, 18 to 26, and 72 to 76 of SEQ ID NO: 2 is substituted with cysteine.
As the PD-L1 protein,
The PD-L1 protein comprises an Ig-V-like domain and an Ig-C-like domain of human PD-L1 protein,
The human PD-L1 protein is SEQ ID NO: 1,
Wherein at least one of the amino acid residues at positions 22 to 25, 36 to 44, 90 to 94, 136 to 145, 154, 185, 196 and 228 to 230 of SEQ ID NO: 1 is substituted with cysteine. L1 protein.
3. The method according to claim 1 or 2,
Wherein the modified PD-L1 protein further comprises an immunoglobulin Fc region.
The method of claim 3,
Wherein said PD-L1 protein and immunoglobulin Fc region are linked to each other through a linker or hinge region.
5. The method of claim 4,
Wherein said immunoglobulin Fc region is an Fc region of a modified immunoglobulin.
6. The method of claim 5,
Wherein the Fc region of the modified immunoglobulin comprises a CH2 domain and a CH3 domain in the N-terminal to C-terminal direction,
Wherein the CH2 domain comprises a portion of the amino acid residue of the CH2 domain of human IgD and human IgG4,
Wherein the CH3 domain comprises a portion of the amino acid residue of the CH3 domain of human IgG4.
A dimeric fusion protein comprising a modified PD-L1 protein,
7. A fusion protein comprising a first monomer and a second monomer selected from the fusion proteins according to any one of claims 1 to 6,
The first monomer and the second monomer have different sequences,
Wherein either one of the cysteine residues of the first monomer and any of the cysteine residues of the second monomer is covalently bonded.
8. The method of claim 7,
Wherein the covalent bond is a disulfide bond.
8. The method of claim 7,
Wherein the lg-V-like domain of said first monomer comprises at least one of the amino acid residues at positions 72 to 76 of SEQ ID NO: 2 and at least one of the amino acid residues at positions 4 to 7 of SEQ ID NO: 2 of the lg- Wherein said amino acid residue is substituted with cysteine.
8. The method of claim 7,
Wherein the PD-L1 protein of the first monomer is one in which at least one amino acid residue in the amino acid residues at positions 136 to 145, 154, 185 and 196 of SEQ ID NO: 1 is replaced by cysteine; And at least one amino acid residue in the amino acid residues 139 to 148 and 228 to 230 of SEQ ID NO: 1 of the PD-L1 protein of said second monomer is cysteine-biased.
11. The method of claim 10,
Wherein at least one amino acid residue in the amino acid residues 90 to 94 of SEQ ID NO: 1 of the PD-L1 protein of the first monomer is substituted with cysteine; And wherein at least one amino acid residue of the amino acid residue at position 22 to 25 of SEQ ID NO: 1 of the PD-L1 protein of said second monomer is substituted with cysteine.
8. The method of claim 7,
One of the CH3 domains of the Fc region of the modified monomer of the first monomer and the modified monomer of the second monomer is knob-shaped, and the other is a knob-into-hole Lt; / RTI &gt; fusion protein.
13. The method of claim 12,
Wherein said knob strain is a dimer fusion protein wherein the amino acid residue at position 152 of SEQ ID NO: 9 is cysteine and the amino acid residue at position 164 is tryptophan.
13. The method of claim 12,
Wherein the amino acid residue at position 147 is SEQ ID NO: 9 is cysteine, the amino acid residue at position 164 is serine, the amino acid residue at position 166 is alanine, and the amino acid residue at position 205 is valine. .
14. A pharmaceutical composition for preventing or treating an immunological disease comprising the fusion protein according to any one of claims 1 to 14.
16. The method of claim 15,
Wherein said immune disease is selected from the group consisting of autoimmune diseases, inflammatory diseases, and graft-rejection diseases of cells, tissues, or organs.
14. A composition for inducing immunostimulation comprising the fusion protein according to any one of claims 1 to 14.
15. A method for the prevention or treatment of an immune disorder comprising administering to a subject a fusion protein according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

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